Pergamon Int. J. Devl Neuroscience, Vol. 13, No. 8, pp. 825-834, 1995 Elsevier Science Ltd

Copyright © 1995 ISDN 0736-S']48(95)~0..4 Printed in Great Britain. All rights reserved

0736-5748/95 $9.50+0.00

NEURONAL AND NON-NEURONAL CATECHOL-O-METHYLTRANSFERASE IN PRIMARY CULTURES

OF RAT BRAIN CELLS

T. K A R H U N E N , * t C. T I L G M A N N , ~ I. U L M A N E N $ and P. P A N U L A t § *Institute of Biomedicine, Department of Anatomy, University of Helsinki, Helsinki, Finland;

tDepartment of Biology, Abo Akademi University, Turku, Finland; ~tOrion Corporation, Orion-Farmos Pharmaceuticals, Orion Research, Helsinki, Finland

(Received 7 March 1995; revised 29 June 1995; accepted 29 June 1995)

Abstract--Previous biochemical and histochemical studies have suggested that catechol- O-methyltransferase (COMT) is a predominantly glial enzyme in the brain. The aim of this work was to study its localization and molecular forms in primary cultures, where cell types can be easily distinguished with specific markers. COMT immunoreactivity was studied in primary astrocytic cultures from newborn rat cerebral cortex, and in neuronal cultures from rat brain from 18-day-old rat embryos using antisera against rat recombinant COMT made in guinea pig. Double-staining studies with specific cell markers to distinguish astrocytes, neurons and oligodendrocytes were performed. COMT immunoreactivity colocalized with a specific oligodendrocyte marker galactocerebroside in cells displaying oligodendrocyte morphology, fiat cells displaying type-1 astrocyte morphology and glial fibrillary acidic protein, in branched cells displaying type-2 astrocyte morphology and in cell bodies of neurons, the processes of which displayed neurofilament immunoreactivity. Western blots detected both soluble 24 kDa and membrane-bound 28-kDa COMT proteins in neuronal and astrocyte cultures. The results suggest that COMT is synthesized by cultured astrocytes, oligodendrocytes and neurons.

Keywords: catecholamines, astrocytes, oligodendrocytes.

In the central nervous system, ca techol -O-methyl t ransferase ( C O M T ) inactivates the catechol- amine neuro t ransmi t te rs dopamine , noradrena l ine and adrenal ine 7,55 by t ransferr ing a methyl g roup f rom S-adenosyl -L-methionine to the catechol substrate. 4 T w o forms of C O M T , the soluble ( S - C O M T ) and m e m b r a n e - b o u n d ( M B - C O M T ) , have been character ized and the funct ional and kinetic proper t ies o f these two forms has been described. 2,18,26,30,42,44,52 Recen t cloning of the genes f rom rat and man made cell-free t ranslat ion studies possible. 5,32,46 The C O M T clones suggest that the m e m b r a n e - b o u n d fo rm of the enzyme is an integral m e m b r a n e prote in and that the catalytic par t of the enzyme is o r ien ted towards the cytoplasmic side o f the membrane . 56 In rat brain, only the 1.9-kb M B - m R N A species can be detected, 49,5° but bo th C O M T forms are found there. 18,42,51 It has been suggested that the soluble C O M T is mainly located in glial cells, whereas the M B - C O M T would be the functionally significant fo rm in neurons. 42,43 Act ivi ty studies reveal that the distr ibution of C O M T in the central ne rvous sys tem is relatively even, and does not follow the d i s t r ibu t ion o f c a t e c h o l a m i n e s o r c a t e c h o l a m i n e synthes iz ing enzymes . 3,6,f° By i m m u n o h i s t o - chemical techniques C O M T has been localized only to non-neurona l cells in the brain. Light microscopic studies with antibodies against isolated C O M T suggest that C O M T resides in lep tomeninges , ependymal cells of the brain ventricles, in epithelial cells of the plexus chor ioideus and glial cells in the rat brain. 23-25,42 SubceUular f ract ionat ion studies and also some lesion studies indicate a possible neurona l localization of this enzyme. 1,2°,35,42

O u r recent studies with ant isera against rat r ecombinan t C O M T prote in suggest tha t C O M T in rat brain is f ound bo th in neu rons and glial cells 24,33 and that bo th fo rms are de tec ted in different

§To whom all correspondence should be addressed at: Abo Akademi University, Department of Biology, Biocity, Tykist6katu 6A, SF-20520 Turku, Finland.

Abbreviations: c-Ara, cytosine-l-[3-D-arabinofuranoside; COMT, catechol-O-methyltransferase; BME, Basal Medium+ Eagle; DMEM, Dulbecco's Modified Eagle medium; FCS, fetal calf serum; GalC, galactocerebroside; GFAP, glial fibrillary acidic protein; HRP, horseradish peroxidase; MB-COMT, membrane-bound catechol-O-methyltransferase; NF, neurofilament; PBS, phosphate-buffered saline; PBS-S, phosphate-buffered saline containing 0.05% saponin; PFA, paraf- ormaldehyde; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; S-COMT, soluble catechol-O- methyltransferase.

825

826 T. Karhunen et al.

parts of the brain, a6 In this study we further examine the expression of COMT in different cell types in cultures enriched with one cell type derived from rat brain. COMT immunoreactivity in different primary cultures was studied using polyclonal antibodies against purified, E. coli-expressed rat recombinant COMT protein. These antibodies detect both soluble 24 kDa and the membrane- bound 28 kDa form of the enzyme. 25,33 Western blots were performed to confirm the presence of COMT both in astrocytes and neurons and also to clarify whether both forms of COMT are found in these cells. Immunoelectron microscopy was used to detect the subcellular localization of COMT.

EXPERIMENTAL PROCEDURES

Antibodies

Polyclonal antibodies against purified E. coli-expressed rat S-CO1VIT 34 were used in this study. The production, characterization and specificity of the COMT antisera have been described earlier.25, 34

Sample preparation for immunoblotting

Cells were homogenized in 10 mM sodium phosphate buffer, pH 7.4, containing 0.2 mM phenylmethylsulfonyl fluoride and the homogenate centrifuged at 2500 rpm for 20 min. The proteins in the supernatant were separated on 10% SDS-PAGE, 28 followed by electrophoretic transfer onto polyvinylidene difluoride membranes (PVDF, Immobilon P, Millipore). 53 Equal amounts of proteins from both astrocytes and neurons were loaded. The filters were treated with a guinea pig antiserum raised against rat recombinant S-COMT protein (1:200 dilution) in PBS/0.1% Tween 20 and 5% (w/v) non-fat dry milk for 1.5 hr at room temperature (RT), 19 followed by peroxidase-conjugated sheep anti-guinea pig Ig (Boehringer-Mannheim, Mannheim, Germany) for 1 hr at RT. 4-Chloro-l-napthol (Sigma, St. Louis, MO) was used as the substrate) 6

Preparation of cultures

To obtain optimal numbers of different cells, three different methods were used to culture brain cells. Astrocyte cultures were derived from the neocortex of newborn rats of Wistar strain. Astrocyte cultures as well as neuronal cultures were derived by modifying the protocol described earlier) 4 Mechanically dissociated tissue was sieved through a 80-~m polyester mesh and suspended in culture medium and spread on poly-L-lysine-treated glass coverslips 36 placed in plastic Petri dishes (Nunc, Copenhagen, Denmark), 50 mm in diameter. The culture medium was Dulbecco's Modified Eagle medium (DMEM, Gibco Grand Island, NY) containing 20% (v/v) fetal calf serum (FCS, Gibco) at the time of plating and 10% (v/v) after three days. The cultures were incubated at 37°C in a water-saturated atmosphere containing 5% CO2 for a minimum of two weeks, after which the cells formed a continuous monolayer.

The type-2 astrocyte cultures were derived from the cortex of one- to two-day-old rats. 17 The cerebral cortices were collected into Basal Medium Eagle (Modified) with Earle's salts (BME, Flow Laboratories, Irvine, Scotland) supplemented with 2 mM L-glutamine, penicillin (250,000 IU/1) and streptomycin (0.5%). The cells were triturated mechanically with a Pasteur-pipette in 2-3 ml of Dispase (neutral protease, Dispase Grade II, Boehringer-Mannheim Bioproducts, Germany) solution (3 mg/ml) in BME and then incubated in this solution for 15 min (37°C). The first suspension of dissociated cells was discarded. The remaining tissue was again treated with 2-3 ml of Dispase solution and the resulting suspension of dissociated cells was collected and combined with 20 ml of culture medium. This procedure was repeated five to six times until only residual tissue remained. The cell suspension was centrifuged (2500 rpm, 10 min) and the cell pellet was resuspended in 20 ml of fresh medium containing 10% FCS. The cell suspension was then seeded in a plastic culture flask. The medium was changed two times a week and the cultivation was continued for two weeks in an atmosphere containing 5% CO2. Prior to shaking, the medium was changed and the flask was incubated about 2 hr to balance the atmosphere in the flask. Then the cap was tightened to retain the 5% C02/95% air atmosphere and the bottle was wrapped in aluminium foil and shaken at fixed speed for 24 hr at room temperature. After shaking the medium with resuspended cells was sieved through a 30-1xm polyester mesh, which is permeable to single

COMT in primary cell cultures from rat brain 827

cells only. The cell suspension was then centrifuged (2500 rpm, 10 min), the cell pellet resuspended in 10 ml fresh medium and seeded on poly-L-lysine-coated glass coverslips placed in plastic culture dishes, 50 nun in diameter. The cells were then cultivated one week in the presence of 10% FCS.

Neuronal cultures were derived from 18-day-old Wistar rat embryos. The whole brain was dissociated in BME and plated as astrocyte cultures. The medium was supplemented with 2 mM L-glutamine, glucose (Sigma) to provide a final concentration of 6 g/l, 250,000 IU/I penicillin and 0.5% streptomycin. The medium was changed first after one day of cultivation and then after six days, the amount of FCS being changed from 20 to 10% (v/v). After 10 days of cultivation the amount of FCS was reduced to 5% (v/v). After six days of cultivation the cultures were treated with 10 -5 M cytosine-l-[3-D arabinofuranoside (c-Ara; Sigma) to suppress the growth of dividing cells. Cultivation was continued up to 16 days.

Immunohistochemistry The indirect immunofluorescent technique was used. The cultures were washed briefly with

0.1 M phosphate-buffered saline (PBS; pH 7.4) and fixed with 4% paraformaldehyde (PFA; Merck, Darmstadt, Germany) dissolved in 0.1 M sodium phosphate buffer, pH 7.4, for 10 min at room temperature and rinsed with PBS 2×10 min. The cultures were treated with PBS containing 0.25% Triton X-100 for 15 min at room temperature. The incubation with the primary COMT antiserum or normal guinea pig serum as a control was carried out in PBS overnight at 4°C. To identify different cell types in cultures, double-staining studies together with COMT antiserum the following antisera against neuron- and glia-specific markers were performed: rabbit anti-glial fibrillary acidic protein (GFAP, Dakopatts, Copenhagen, Denmark) specific for astrocytes, mouse monoclonal anti-neurofilament (NF) 58 specific for neurons and mouse monoclonal anti-galacto- cerebrocide (GalC, Boehringer-Mannheim Bioproducts) specific for oligodendrocytes. 4° The antibodies were visualized with corresponding fluorescein- and rhodamine-labelled anti-IgGs (Dakopatts, diluted 1:40; and Cappel, West Chester, PA, diluted 1:100) for 1 hr at room temperature. Coverslips with the cells were washed with PBS 2×10 min and embedded in a mixture of glycerol and PBS (1:1, v/v). The specimens were examined under a Nikon microscope equipped with appropriate filter system and photographed with a Nikon 801 camera on Kodak T-max film (400 ASA).

Immunoelectron microscopy

Cells were fixed with 4% PFA containing 0.5% glutaraldehyde (Fluka, Buchs, Switzerland) in 0.1 M sodium phosphate buffer, pH 7.4, for 30 min at room temperature. Cells were incubated in primary antisera or normal guinea pig serum as above, except that dilutions and all washing steps were performed in PBS- 0.05% saponin (PBS-S). COMT immunoreactivity was demonstrated with peroxidase-conjugated biotin-avidin method (Vector Lab., CA). After primary antiserum, cells were washed and incubated in biotinylated secondary antiserum diluted 1:1000. Cells were again washed and incubated in avidin coupled to HRP (Vector), diluted 1:1000. Peroxidase reaction was detected with 3,3'-diaminobenzidine (Sigma), 50 mg/100 ml Tris-HC1 buffer, pH 7.6, containing 0.001% 14202. Cells were post-fixed with 1% osmium tetroxide for 1 hr at room temperature and dehydrated through a graded ethanol series. After dehydration the cells were embedded in Epon. Ultrathin sections were cut and examined under JEOL SX100 electron microscope without post-staining and using low acceleration voltage (40 kV).

RESULTS

Western blotting analyses on rat neuronal and glial primary cultures indicated both the 28-kDa MB- and 24-kDa S-COMT forms to be present in these cells (Fig. 1). Weak bands larger than 28 kDa, apparently not related to COMT were detected (lanes 2 and 3). Double immunostaining of the astroglial-enriched cultures, prepared from cortical hemispheres of newborn rats, with polyclonal antibodies against rat purified COMT and glial fibrillary acidic protein located COMT immunoreactivity to the same cells with GFAP (Fig. 2A, B). COMT immunofluorescence was distributed evenly in the cytoplasm. Figure 2C displays a phase-contrast view of this culture showing typical flat, polygonal type-1 astrocytes. Figure 3A and B shows immunostaining of astrocyte

828 T. Karhunen et al.

1 2 3

2'

Fig. 1. Immunoblot analysis on COMT in different primary cell cultures. Proteins were separated and detected as described in Experimental Procedures. Lane 1: in-vitro translated S- and MB-COMT (24 and 28 kDa, respectively). Lane 2 represents astroglial and lane 3 neuronal primary culture. In astroglial culture, the S-COMT slightly predominates over the MB-form, whereas the two COMT forms seem to appear in about equal amounts in neurons. Glial cells, and to a minor degree also neurons, show some cross-reacting

bands to other proteins.

cultures with COMT antiserum and normal guinea pig serum as a control, respectively. No immunoreact ion could be seen in the control cultures. Cells displaying the stellate morphology, a round cell body and radial processes, of type-2 astrocytes, in cultures where cells of this type are enriched, were intensely immunoreactive for COMT (Fig. 3C).

Single cells in type-2 astrocyte cultures expressed galactocerebroside (GalC), the major glycolipid of myelin, a specific cell-surface marker used for oligodendrocytes in culture. COMT immunore- activity (Fig. 2D) was detected in the cells expressing GalC immunoreactivity (Fig. 2E). The major morphological features of oligodendrocytes, small spherical perikarya and numerous fine, branched cell processes are seen in Fig. 2F. Also those few small, process-bearing cells growing on the top of the layer of flat cells in type-1 astroglial culture expressing GalC were immunoreactive for COMT (not shown). COMT immunoreactivity in these cells was more intense than in flat type-1 astrocytes, which formed the major cell population in these cultures.

COMT immunoreactivity was prominent also in neuronal cells. In neuronal cell cultures derived from 18-day-old embryonic whole brain there were COMT immunoreactive cells which expressed NF immunoreactivity (Fig. 2G and H, respectively). There were also COMT immunoreactive cells displaying neuron-like morphology without NF immunoreactivity (Fig. 2G, H and I). The identity of these cells remains unclear.

In primary type-1 astrocytic cultures immunoelectron microscopy revealed COMT immunoreac- tivity in the cytoplasm and in association with tubular structures of astrocytes (Fig. 4A, B). Patchy accumulation of reaction product was also observed along the nuclear membrane (Fig. 4B). The reaction product was evenly distributed in the whole cytoplasm and reached the plasma membrane. The nucleus was devoid of reaction (Fig. 4A, B).

DISCUSSION

COMT has been localized immunocytochemically with antisera against purified natural enzyme only to non-neuronal cells in the brain. 21-23 Our recent study using specific antibodies against purified, E. coli-expressed recombinant COMT protein revealed COMT immunoreactivity in glial

C O M T in primary cell cultures f rom rat brain 829

Fig. 2. (A) Double staining of primary astroglial cell culture with COMT antiserum and (B) glial fibrillary acidic protein antiserum located COMT immunoreactivity to astrocytes. (C) Corresponding phase contrast micrograph view of the culture revealed a continuous layer of flat, polygonal cells. (D)-(E) COMT immunoreactivity (D) in cells which were judged to be oligodendrocytes by a specific cell-surface marker galactocerebrocide antiserum (E). (F) A corresponding phase contrast view revealed a group of these branched cells. (G-H) In neuronal primary culture there were COMT immunoreactive cells (G) which also showed neurofilament immunoreactivity (H; short arrow). Part of the cells showing immunoreactivity for COMT (G) were not, however, neurofilament immunoreactive (H; long arrow). (I) A corresponding phase contrast micrograph of the neuronal culture in. Bars: 50 ixm in (A), (B), (C), (G), (H) and (I) and

100 ixm in (n), (E) and (F).

|3-8-C

830 T. Karhunen et al.

Fig. 3. (A) COMT immunoreactivity in type-1 astrocytes. (B) No reaction was seen when cultures were incubated with the control antiserum. (C) Stellate cells in type-2 astrocyte culture showed bright

immunoreactivity for COMT. Bars: 50 tzm in (A) and 100 I*m in (B) and (C).

cells and also fine, punctate COMT immunoreactivity in the striatal neuropil and in the neuropil of cortical areas of the rat brain was prominent. It was impossible at light microscopic level to determine the cell type responsible for COMT synthesis, because the reaction was most prominent in processes of the cells. In spinal ganglia a population of sensory neurons contained COMT immunoreactivity. 25 In this study expression of COMT immunoreactivity in different cultured cell types derived from rat brain was studied. Both astrocytes and neurons, as well as oligodendrocytes were immunoreactive for COMT. Furthermore, both forms of the enzyme were present in astroglial and neuronal cultures.

It is believed that type-2 astrocytes and oligodendrocytes are derived from a common progenitor cell type 8,31,39 whereas type-1 astrocytes originate from a separate macroglial lineage. 38 In cell cultures the bipotential O-2A (oligodendrocyte-type-2 astrocyte) progenitor cells are capable of differentiating either to oligodendrocytes or type-2 astrocytes depending on the FCS content of the medium. In the presence of FCS greater part of these progenitor cells differentiate to type-2 astrocytes, but cells expressing oligodendrocyte properties are also present. 39 In the present study O-2A progenitor cells were isolated from astroglial cultures by vigorous shaking and grown in the presence of 10% FCS and characterized with anti-Galc. Both negative and immunoreactive cells for GalC appeared. Cells negative for GalC immunoreactivity were con- sidered to be type-2 astrocytes also according to their stellate morphology. Both cell types showed bright immunofluorescence for COMT.

A possible role suggested for COMT in the brain is to form barriers between different compartments of catechol compounds and also to prevent the diffusion of catechols escaping from the catecholaminergic neurons. 23 The distribution of COMT in tl~e ependymal cells of ventricles and the epithelial cells of the choroid plexus, 21-23,29 in cerebrovascular endothelial cells, 27,48 and widely in astrocytes and oligodendrocytes 23 could provide this kind of limitation.

COMT activity has been previously characterized in primary cultures of astrocytes 13J5 and localized immunohistochemically in cerebrovascular endothelial and smooth muscle cells. 48 COMT activity has also been reported to be present in strains of rodent astrocytoma 9,47 as well as in rat neuroblastoma cells. 9 The results of this study indicate that neurons and oligodendrocytes also contain COMT in vitro, and that both the soluble and the membrane-bound forms of the enzyme are present in neuronal and non-neuronal primary cultures. The COMT activity has been shown to be higher in primary astrocytic cultures than in adult rat hemispheres. 13J5 COMT activity increases during cultivation and the enzyme activity in two-week-old cultures is 1.5 times higher than activity in homogenates of adult rat brain hemispheres. The more pronounced presence of COMT immunoreactivity in cultured cells in comparison to previous studies with brain tissue sections 25 may be explained by the presence of low levels of COMT in adult rat brain sections. It is possible that expression of COMT is upregulated in culture conditions also in cells which in situ express low amounts of the enzyme. It is unlikely that the cross-reacting bands detected interfere with the immunostaining, because such bands can also be detected in the brain tissue, 25 and preabsorption

COMT in primary cell cultures from rat brain 831

Fig. 4. (A) An overall pre-embedding immunoelectron micrographic view of a COMT immunoreactive astrocyte in a primary culture. Reaction end product was seen throughout the cytoplasm and in tubular structures (arrows). No prominent immunoreaction was seen in the plasma membrane. (B) A greater magnification of an immunoreactive astrocyte. Immunoreaction in the cytoplasm was prominent and patchy accumulation of the reaction product in nuclear membrane was also seen (arrows). Bars: 5 ixm in

(A) and 500 nm in (B). m, mitochondria; N, nucleus.

of the antiserum with purified recombinant protein completely abolished the reaction. However, further studies to elucidate this point with new antisera, and in-situ hybridization studies to detect COMT mRNA would verify the result.

Indirect evidence also supports the presence of COMT enzyme in neurons. In cell fractionation studies COMT activity has been found in the fractions which contain the nerve terminals. 1 Lesion studies, after identifying the different COMT forms, support the localization of MB-COMT post-synaptically in neurons 20,35,4a whereas there is no evidence of presynaptic localization of COMT. Also our recent finding of the localization of COMT in astrocyte processes around synapses and in post-synaptic dendrites in rat brain 24 suggests that presynaptic COMT is not important. In rat tissues the membrane-associated form of COMT comprises a minor part of the total enzyme

832 T. Karhunen et al.

activity. 11,12,18,33,42,45 However, the physiological significance of this form in the O-methylation of catecholamine neurotransmitters in neuronal cells in the central nervous system has been suggested to be more important than that of the soluble form. 30,43,44

Regulation of COMT synthesis in cultures of characterized cell types and fine structural localization of the two forms in neurons and glia are now important for better understanding of the functional role of COMT. The relation of COMT to the cell membrane is an essential question to solve. It is especially important to find out if MB-COMT occurs in neurons in the post-synaptic membrane as has been speculated. 44 Our recent immunoelectron microscopic study gives some evidence on that. 24 The presence of COMT in plasma membranes of adipocytes 54 and liver cells, 2,41 or in outer mitochondrial membrane 11 has been suggested. More recent cell fractionation and Percoll gradient studies of the rat brain have placed MB-COMT to plasma membranes and/or rough endoplasmic reticulum rather than to mitochondrial membrane. 51 That MB-COMT is located in intracellular membranes and in rough endoplasmic reticulum is supported by the latest immunoh- istochemical studies on mammalian cells expressing recombinant MB-COMT. Double staining of these cells with antisera against recombinant COMT and proteins of rough endoplasmic reticulum colocalized these two signals. 33

Pre-embedding immunoelectron microscopy of cultured astrocytes revealed cytoplasmic reac- tion, and a patchy distribution of the reaction product was also seen in the nuclear membrane. Although no apparent accumulation of reaction product along the cell membrane was observed, the nature and distribution of DAB reaction product did not allow reliable analysis of membrane localization of COMT. Our attempts to localize COMT with post-embedding immunogold methods have been hampered by the sensitivity of COMT proteins to polymerization procedures (unpub- lished results). Therefore, an immunogold procedure applied to frozen ultrathin sections may be needed to clarify the more detailed subcellular localization of COMT in neurons and glial cells.

Acknowledgements--This study was supported by the Medical Research Council of the Academy of Finland, The Sigrid Juselius Foundation and Research and Science Foundation of Farmos.

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